Corrections to "A Sub-Pixel Accurate Quantification of Joint Space Narrowing Progression in Rheumatoid Arthritis".

Presents corrections to the article "A Sub-Pixel Accurate Quantification of Joint Space Narrowing Progression in Rheumatoid Arthritis".

Clinical features of sleep apnea syndrome and cognitive impairment in multiple sclerosis.

BACKGROUND: Complications of obstructive sleep apnea (OSA) have been reported in patients with multiple sclerosis (MS). Patients with sleep apnea syndrome (SAS) due to OSA also show cognitive decline, with similar clinical characteristics to that manifested in MS. SAS due to OSA is a treatable condition, and the associated cognitive decline is expected to improve. This study investigates clinical features of SAS in people living with MS and contribute to improve cognitive dysfunction of MS. METHODS: A case-control study was conducted. Cognitive functions were evaluated by the Symbol Digit Modalities Test (SDMT) and the Paced Auditory Serial Addition Test 2 (PASAT-2) and 3 (PASAT-3). The Respiratory Event Index (REI) was measured using Out of Center Sleep Testing (OCST). We defined subjects with REI ≥ 5 as OSA and divided participants into two groups with or without SAS due to OSA. Cognitive and respiratory characteristics were statistically compared between patients with MS and healthy controls. RESULTS: We enrolled 67 people living with MS and 31 age- and sex-matched controls. OCST detected OSA in people living with MS and controls, and the prevalence rates were 28.4 % and 25.8 %, respectively. REI values (5.2 ± 7.9 vs 3.9 ± 5.2, p = 0.509) and number of participants with REI ≥ 5 (19 vs 8, p = 0.793) were similar between the MS and control group. The SDMT, PASAT-2, and PASAT-3 scores were significantly lower in the MS group than the control group (p < 0.001, p = 0.001, and p < 0.001, respectively). The interaction effect of MS and SAS on cognitive function was not significant in the SDMT (p = 0.078), but in the PASAT-2 (p = 0.043) and PASAT-3 (p = 0.020). CONCLUSION: This study revealed the prevalence rates of SAS in Japanese people living with MS and the usefulness of OCST for detection of SAS. This study also revealed that concomitant SAS can facilitate cognitive decline in people living with MS. These findings suggest that an appropriate intervention for OSA can be beneficial for people living with MS with cognitive decline.

A Sub-Pixel Accurate Quantification of Joint Space Narrowing Progression in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily affects peripheral synovial joints, like fingers, wrists and feet. Radiology plays a critical role in the diagnosis and monitoring of RA. Limited by the current spatial resolution of radiographic imaging, joint space narrowing (JSN) progression of RA for the same reason above can be less than one pixel per year with universal spatial resolution. Insensitive monitoring of JSN can hinder the radiologist/rheumatologist from making a proper and timely clinical judgment. In this paper, we propose a novel and sensitive method that we call partial image phase-only correlation which aims to automatically quantify JSN progression in the early RA. The majority of the current literature utilizes the mean error, root-mean-square deviation and standard deviation to report the accuracy at pixel level. Our work measures JSN progression between a baseline and its follow-up finger joint images by using the phase spectrum in the frequency domain. Using this study, the mean error can be reduced to 0.0130 mm when applied to phantom radiographs with ground truth, and 0.0519 mm standard deviation for clinical radiography. With the sub-pixel accuracy far beyond usual manual measurements, we are optimistic that the proposed work is a promising scheme for automatically quantifying JSN progression.

Fully automatic software for detecting radiographic joint space narrowing progression in rheumatoid arthritis: phantom study and comparison with visual assessment.

PURPOSE: We have developed an in-house software equipped with partial image phase-only correlation (PIPOC) which can automatically quantify radiographic joint space narrowing (JSN) progression. The purpose of this study was to evaluate the software in phantom and clinical assessments. MATERIALS AND METHODS: In the phantom assessment, the software's performance on radiographic images was compared to the joint space width (JSW) difference using a micrometer as ground truth. A phantom simulating a finger joint was scanned underwater. In the clinical assessment, 15 RA patients were included. The software measured the radiological progression of the finger joints between baseline and the 52nd week. The cases were also evaluated with the Genant-modified Sharp score (GSS), a conventional visual scoring method. We also quantitatively assessed these joints' synovial vascularity (SV) on power Doppler ultrasonography (0, 8, 20 and 52 weeks). RESULTS: In the phantom assessment, the PIPOC software could detect changes in JSN with a smallest detectable difference of 0.044 mm at 0.1 mm intervals. In the clinical assessment, the JSW change of the joints with GSS progression detected by the software was significantly greater than those without GSS progression (p = 0.004). The JSW change of joints with positive SV at baseline was significantly higher than those with negative SV (p = 0.024). CONCLUSION: Our in-house software equipped with PIPOC can automatically and quantitatively detect slight radiographic changes of JSW in clinically inactive RA patients.

Direct Rub Inoculation of Fungal Flora Changes Fatty Acid Composition and Volatile Flavors in Dry-Aged Beef: A Preliminary Study.

Here, we established a method to produce dry-aged beef (DAB) by rub inoculation with fungal flora on the prepared DAB surface. Portions of Holstein steers' rumps were prepared by direct rub inoculation of fungal flora or without treatment (conventional DAB) and dry-aged for 26 days in an aging room at 2.9 °C and 90% relative humidity. We compared the fungal covering and meat quality, including fatty acid composition and volatile aromatic compounds, of fungal-inoculated DAB with those of the conventional DAB. The fungal-inoculated DAB was almost entirely covered with white mold, in contrast to the conventional DAB. Moreover, the proportion of oleic acid and the concentration of nine volatile compounds significantly increased in the raw meat of fungal-inoculated DAB compared with those in the conventional DAB (p < 0.05). These results suggested that direct rub inoculation of fungal flora from prepared DAB may accelerate DAB production and efficiently enhance the "melt-in-the-mouth" feeling and flavors of DAB.

Change of clopyralid concentration in recycled beef cattle compost.

Composting of beef cattle manure using sawdust or recycled compost as a bulking agent was investigated for the management of clopyralid risk, such as changes in the clopyralid concentration and the mechanism of clopyralid accumulation caused by recycled compost. These raw materials were composted with laboratory equipment, which was controlled at 60℃ after the temperature peak by autothermal composting. Clopyralid concentration did not changed during composting in the thermophilic phase; on the contrary, it increased because clopyralid accumulated in compost when recycled compost was used repeatedly as a bulking agent. The clopyralid accumulation ratio (ratio of clopyralid concentration to that in the first compost) could be explained by a model using a recurrence formula, and a correlation existed between the calculated (model) accumulation ratios and measured accumulation ratios (R2  = 0.78). Using this model, the excessive accumulation of clopyralid could be controlled when using recycled compost as a bulking agent with lower moisture content or when part of the recycled compost was replaced by another bulking agent, even if recycled compost had high moisture content. In future work, the model and its considerations should be verified in a field test.

Quantification of Joint Space Width Difference on Radiography Via Phase-Only Correlation (POC) Analysis: a Phantom Study Comparing with Various Tomographical Modalities Using Conventional Margin-Contouring.

Several visual scoring methods are currently used to assess progression of rheumatoid arthritis (RA) on radiography. However, they are limited by its subjectivity and insufficient sensitivity. We have developed an original measurement system which uses a technique called phase-only correlation (POC). The purpose of this study is to validate the system by using a phantom simulating the joint of RA patients.A micrometer measurement apparatus that can adjust arbitrary joint space width (JSW) in a phantom joint was developed to define true JSW. The phantom was scanned with radiography, 320 multi detector CT (MDCT), high-resolution peripheral quantitative CT (HR-pQCT), cone beam CT (CBCT), and tomosynthesis. The width was adjusted to the average size of a women's metacarpophalangeal joint, from 1.2 to 2.2 mm with increments of 0.1 mm and 0.01 mm. Radiographical images were analyzed by the POC-based system and manual method, and images from various tomographical modalities were measured via the automatic margin detection method. Correlation coefficients between true JSW difference and measured JSW difference were all strong at 0.1 mm intervals with radiography (POC-based system and manual method), CBCT, 320MDCT, HR-pQCT, and tomosynthesis. At 0.01 mm intervals, radiography (POC-based system), 320MDCT, and HR-pQCT had strong correlations, while radiography (manual method) and CBCT had low correlations, and tomosynthesis had no statistically significant correlation. The smallest detectable changes for radiography (POC-based system), radiography (manual method), 320MDCT, HR-pQCT, CBCT, and tomosynthesis were 0.020 mm, 0.041 mm, 0.076 mm, 0.077 mm, 0.057 mm, and 0.087 mm, respectively. We conclude that radiography analyzed with the POC-based system might sensitively detect minute joint space changes of the finger joint.

Comparative study between lactose-silk fibroin conjugates and extracellular matrices as a substrate for the culture of human induced pluripotent stem cell-derived hepatocytes.

BACKGROUND: Human induced pluripotent stem cell (hiPSC)-derived hepatocytes are an attractive alternative cell source to primary human hepatocytes for tissue regeneration. OBJECTIVES: This study presents an application of lactose-silk fibroin conjugates (Lac-CY-SF) bearing 𝛽-galactose residues as a substrate for culture of hiPSC-derived hepatocytes. A comparison of hiPSC-derived hepatocytes cultured on three different substrates; Lac-CY-SF conjugates, Matrigel and type I collagen was performed. METHODS: Cell morphology, viability, maturation and albumin secretory function were assessed by phase-contrast microscopy, tetrazolium-based colorimetric assay, immunofluorescence staining and enzyme-linked immunosorbent assay. RESULTS: Morphological characteristics of the cells cultured on the conjugates resembled those on Matrigel throughout the 6-day culture period. The number of viable cells cultured on the conjugates was comparable to that on Matrigel at day 2 and 6. The protein expression of mature hepatocyte markers, asialoglycoprotein receptor 1 and albumin, by the cells cultured on the conjugates resembled that by the cells cultured on collagen at day 2 and 6. Albumin secretory function per cell cultured on the conjugates was higher than that on collagen and comparable to that on Matrigel. CONCLUSIONS: These limited results suggest that Lac-CY-SF conjugates may be as useful as Matrigel and collagen for cultivation of hiPSC-derived hepatocytes.

Detection of Fine Radiographic Progression in Finger Joint Space Narrowing Beyond Human Eyes: Phantom Experiment and Clinical Study with Rheumatoid Arthritis Patients.

The visual assessment of joint space narrowing (JSN) on radiographs of rheumatoid arthritis (RA) patients such as the Genant-modified Sharp score (GSS) is widely accepted but limited by its subjectivity and insufficient sensitivity. We developed a software application which can assess JSN quantitatively using a temporal subtraction technique for radiographs, in which the chronological change in JSN between two radiographs was defined as the joint space difference index (JSDI). The aim of this study is to prove the superiority of the software in terms of detecting fine radiographic progression in finger JSN over human observers. A micrometer measurement apparatus that can adjust arbitrary joint space width (JSW) in a phantom joint was developed to define true JSW. We compared the smallest detectable changes in JSW between the JSDI and visual assessment using phantom images. In a clinical study, 222 finger joints without interval score change on GSS in 15 RA patients were examined. We compared the JSDI between joints with and without synovial vascularity (SV) on power Doppler ultrasonography during the follow-up period. True JSW difference was correlated with JSDI for JSW differences ranging from 0.10 to 1.00 mm at increments of 0.10 mm (R2 = 0.986 and P < 0.001). Rheumatologists were difficult to detect JSW difference of 0.30 mm or less. The JSDI of finger joints with SV was significantly higher than those without SV (P = 0.030). The software can detect fine differences in JSW that are visually unrecognizable.

A versatile strategy for the synthesis and mechanical property manipulation of networked biodegradable polymeric materials composed of well-defined alternating hard and soft domains.

The present paper proposes a versatile strategy for the synthesis and mechanical property manipulation of networked biodegradable polymeric materials composed of well-defined alternating soft and hard domains. As an example of the strategy, we selected biodegradable poly(l-lactide) (PLLA) and poly(ε-caprolactone) (PCL) as the hard and soft components, respectively, and synthesized networked biodegradable polymeric materials composed of well-defined alternating PLLA and PCL domains with different l-lactide (LLA) unit contents via crosslinking of well-defined four-armed diblock copolymers of PLLA and PCL (4-C-L). The strategy reported here, which is also applicable to non-biodegradable polymeric materials, successfully facilitated the synthesis of the networked biodegradable materials composed of alternating hard and soft domains and their mechanical properties of the synthesized materials were largely manipulated by the LLA unit contents of the precursor four-armed diblock 4-C-L copolymers. Moreover, the crystallization behavior and thermal properties of 4-C-L copolymers before and after crosslinking were investigated and discussed.

Tomosynthesis can facilitate accurate measurement of joint space width under the condition of the oblique incidence of X-rays in patients with rheumatoid arthritis.

OBJECTIVE: Accurate evaluation of joint space width (JSW) is important in the assessment of rheumatoid arthritis (RA). In clinical radiography of bilateral hands, the oblique incidence of X-rays is unavoidable, which may cause perceptional or measurement error of JSW. The objective of this study was to examine whether tomosynthesis, a recently developed modality, can facilitate a more accurate evaluation of JSW than radiography under the condition of oblique incidence of X-rays. METHODS: We investigated quantitative errors derived from the oblique incidence of X-rays by imaging phantoms simulating various finger joint spaces using radiographs and tomosynthesis images. We then compared the qualitative results of the modified total Sharp score of a total of 320 joints from 20 patients with RA between these modalities. RESULTS: A quantitative error was prominent when the location of the phantom was shifted along the JSW direction. Modified total Sharp scores of tomosynthesis images were significantly higher than those of radiography, that is to say JSW was regarded as narrower in tomosynthesis than in radiography when finger joints were located where the oblique incidence of X-rays is expected in the JSW direction. CONCLUSION: Tomosynthesis can facilitate accurate evaluation of JSW in finger joints of patients with RA, even with oblique incidence of X-rays. ADVANCES IN KNOWLEDGE: Accurate evaluation of JSW is necessary for the management of patients with RA. Through phantom and clinical studies, we demonstrate that tomosynthesis may achieve more accurate evaluation of JSW.

Detection and measurement of rheumatoid bone and joint lesions of fingers by tomosynthesis: a phantom study for reconstruction filter setting optimization.

Rheumatoid arthritis (RA) is a systemic disease that is caused by autoimmunity. RA causes synovial proliferation, which may result in bone erosion and joint space narrowing in the affected joint. Tomosynthesis is a promising modality which may detect early bone lesions such as small bone erosion and slight joint space narrowing. Nevertheless, so far, the optimal reconstruction filter for detection of early bone lesions of fingers on tomosynthesis has not yet been known. Our purpose in this study was to determine an optimal reconstruction filter setting by using a bone phantom. We obtained images of a cylindrical phantom with holes simulating bone erosions (diameters of 0.6, 0.8, 1.0, 1.2, and 1.4 mm) and joint spaces by aligning two phantoms (space widths from 0.5 to 5.0 mm with 0.5 mm intervals), examining six reconstruction filters by using tomosynthesis. We carried out an accuracy test of the bone erosion size and joint space width, done by one radiological technologist, and a test to assess the visibility of bone erosion, done by five radiological technologists. No statistically significant difference was observed in the measured bone erosion size and joint space width among all of the reconstruction filters. In the visibility assessment test, reconstruction filters of Thickness+- and Thickness-- were among the best statistically in all characteristics except the signal-to-noise ratio. The Thickness+- and Thickness-- reconstruction filter may be optimal for evaluation of RA bone lesions of small joints in tomosynthesis.

In situ cross-linked electrospun fiber scaffold of collagen for fabricating cell-dense muscle tissue.

Engineered muscle tissues used as transplant tissues in regenerative medicine should have a three-dimensional and cell-dense structure like native tissue. For fabricating a 3D cell-dense muscle tissue from myoblasts, we proposed the electrospun type I collagen microfiber scaffold of the string-shape like a harp. The microfibers were oriented in the same direction to allow the myoblasts to align, and were strung at low density with micrometer intervals to create space for the cells to occupy. To realize this shape of the scaffold, we employed in situ cross-linking during electrospinning process for the first time to collagen fibers. The collagen microfibers in situ cross-linked with glutaraldehyde stably existed in the aqueous media and completely retained the original shape to save the spaces between the fibers for over 14 days. On the contrary, the conventional cross-linking method by exposure to a glutaraldehyde aqueous solution vapor partially dissolved and damaged the fiber to lose a low-density shape of the scaffold. Myoblasts could penetrate into the interior of the in situ cross-linked string-shaped scaffold and form the cell-dense muscle tissues. Histochemical analysis showed the total area occupied by the cells in the cross section of the tissue was approximately 73 %. Furthermore, the resulting muscle tissue fabricated from primary myoblasts showed typical sarcomeric cross-striations and the entire tissue continuously pulsated by autonomous contraction. Together with the in situ cross-linking, the string-shaped scaffold provides an efficient methodology to fabricate a cell-dense 3D muscle tissue, which could be applied in regenerative medicine in future.

Genetic shift in local rice populations during rice breeding programs in the northern limit of rice cultivation in the world.

The rapid accumulation of pre-existing mutations may play major roles in the establishment and shaping of adaptability for local regions in current rice breeding programs. The cultivated rice, Oryza sativa L., which originated from tropical regions, is now grown worldwide due to the concerted efforts of breeding programs. However, the process of establishing local populations and their origins remain unclear. In the present study, we characterized DNA polymorphisms in the rice variety KITAAKE from Hokkaido, one of the northern limits of rice cultivation in the world. Indel polymorphisms were attributed to transposable element-like insertions, tandem duplications, and non-TE deletions as the original mutation events in the NIPPONBARE and KITAAKE genomes. The allele frequencies of the KITAAKE alleles markedly shifted to the current variety types among the local population from Hokkaido in the last two decades. The KITAAKE alleles widely distributed throughout wild rice and cultivated rice over the world. These have accumulated in the local population from Hokkaido via Japanese landraces as the ancestral population of Hokkaido. These results strongly suggested that combinations of pre-existing mutations played a role in the establishment of adaptability. This approach using the re-sequencing of local varieties in unique environmental conditions will be useful as a genetic resource in plant breeding programs in local regions.

High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes.

Levan, a type of fructan, is an oligomer or polymer with mainly a ß(2,6)-linked fructose chain attached to sucrose. We introduced two timothy genes, PpFT1 and PpFT2, coding for two homologous sucrose:fructan 6-fructosyltransferases into sugar beet. Sugar beet produces a high concentration of sucrose, a starting substrate in fructan synthesis, in the root. Among transgenic T1 lines, we obtained sugar beet transformants that accumulated large amounts of ß(2,6)-linked levans (about 20 to 75mgg(-1) FW) in the roots. The transformed sugar beet plants possessing PpFT1 or PpFT2 produced linear levans with different degrees of polymerization (DP). Namely, the PpFT1 transformants accumulated mainly high DP levans including those with DP>40, while the PpFT2 transformants accumulated levans with DP between 3 and 40. Chromatograms showed that PpFT2 produces pure ß(2,6)-linked linear levans compared with fructans synthesized by PpFT1. These levans belong to the high DP class of plant fructans, but have much shorter DP than that of levans generally produced by microorganisms.

Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense).

Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0°C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to -3°C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering.

Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties.

Fructans can act as cryoprotectants and contribute to freezing tolerance in plant species, such as in members of the grass subfamily Pooideae that includes Triticeae species and forage grasses. To elucidate the relationship of freezing tolerance, carbohydrate composition and degree of polymerization (DP) of fructans, we generated transgenic plants in the model grass species Brachypodium distachyon that expressed cDNAs for sucrose:fructan 6-fructosyltransferases (6-SFTs) with different enzymatic properties: one cDNA encoded PpFT1 from timothy grass (Phleum pratense), an enzyme that produces high-DP levans; a second cDNA encoded wft1 from wheat (Triticum aestivum), an enzyme that produces low-DP levans. Transgenic lines expressing PpFT1 and wft1 showed retarded growth; this effect was particularly notable in the PpFT1 transgenic lines. When grown at 22 °C, both types of transgenic line showed little or no accumulation of fructans. However, after a cold treatment, wft1 transgenic plants accumulated fructans with DP = 3-40, whereas PpFT1 transgenic plants accumulated fructans with higher DPs (20 to the separation limit). The different compositions of the accumulated fructans in the two types of transgenic line were correlated with the differences in the enzymatic properties of the overexpressed 6-SFTs. Transgenic lines expressing PpFT1 accumulated greater amounts of mono- and disaccharides than wild type and wft1 expressing lines. Examination of leaf blades showed that after cold acclimation, PpFT1 overexpression increased tolerance to freezing; by contrast, the freezing tolerance of the wft1 expressing lines was the same as that of wild type plants. These results provide new insights into the relationship of the composition of water-soluble carbohydrates and the DP of fructans to freezing tolerance in plants.

High-dose argatroban therapy for stroke: novel treatment for delayed treatment and the recanalization mechanism.

BACKGROUND: There has been little effective treatment in patients with cerebral infarction at >24 hours after onset. We assessed the effects of high-dose argatroban therapy in delayed administration, and investigated the mechanism based on our clinical findings. METHODS: Argatroban 30 mg was first administered for 15 minutes intravenously, and then 90 mg for 60 minutes followed by 60 mg for 60 minutes were infused continuously. The change of vascular obstruction caused by the treatment was assessed with magnetic resonance angiography. RESULTS: In 4 patients studied, high-dose argatroban resulted in 100% recanalization of occluded vessels (5/5), even though argatroban was administrated >24 hours after onset. On the other hand, when an inadequate dose of argatroban was administered, a hemorrhage was identified. This supports our hypothesis that high-dose argatroban promotes recanalization by deactivating thrombin and exerting an anticoagulant effect on the vascular endothelium. CONCLUSIONS: High-dose argatroban is an effective treatment for cerebral infarction and offers a novel therapeutic approach for delayed hospitalized patients at >24 hours after onset. Additional studies are necessary to identify the cellular and molecular mechanisms and determine the adequate dose in order to reduce risks of complication.

Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.).

Variation in the structures of plant fructans and their degree of polymerization (DP) can be explained as the result of diverse combinations of fructosyltransferases (FTs) with different properties. Although FT genes have been isolated in a range of plant species, sucrose:fructan 6-fructosyltransferase (6-SFT) cDNAs have only been functionally characterized in a few species such as wheat. A novel FT cDNA possessing 6-SFT activity has been identified and characterized from the temperate forage grass, timothy (Phleum pratense L.). The cDNA of an FT homolog, PpFT1, was isolated from cold-acclimated timothy. A recombinant PpFT1 protein expressed in Pichia pastoris showed 6-SFT/sucrose:sucrose 1-fructosyltransferase (1-SST) activity and produced linear beta(2,6)-linked levans from sucrose with higher DPs than present in graminans formed in vitro by wheat 6-SFT (Wft1). PpFT1 and Wft1 showed remarkably different acceptor substrate specificities: PpFT1 had high affinity for 6-kestotriose to produce levans and low affinity for 1-kestotriose, whereas Wft1 preferentially used 1-kestotriose as an acceptor. The affinity of the PpFT1 recombinant enzyme for sucrose as a substrate was lower than that of the Wft1 recombinant enzyme. It is also confirmed that timothy seedlings had elevated levels of PpFT1 transcripts during the accumulation of fructans under high sucrose and cold conditions. Our results suggest that PpFT1 is a novel cDNA with unique enzymatic properties that differ from those of previously cloned plant 6-SFTs, and is involved in the synthesis of highly polymerized levans in timothy.

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information.

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding.